The <named-content content-type="genus-species">Enterococcus faecalis</named-content> EbpA Pilus Protein: Attenuation of Expression, Biofilm Formation, and Adherence to Fibrinogen Start with the Rare Initiation Codon ATT

ABSTRACT The endocarditis and biofilm-associated pili (Ebp) are important in Enterococcus faecalis pathogenesis, and the pilus tip, EbpA, has been shown to play a major role in pilus biogenesis, biofilm formation, and experimental infections. Based on in silico analyses, we previously predicted that...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Maria Camila Montealegre, Sabina Leanti La Rosa, Jung Hyeob Roh, Barrett R. Harvey, Barbara E. Murray
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2015
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/3df68031778a4f8fbdf7fb021cbe20af
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:3df68031778a4f8fbdf7fb021cbe20af
record_format	dspace
spelling	oai:doaj.org-article:3df68031778a4f8fbdf7fb021cbe20af2021-11-15T15:49:03ZThe <named-content content-type="genus-species">Enterococcus faecalis</named-content> EbpA Pilus Protein: Attenuation of Expression, Biofilm Formation, and Adherence to Fibrinogen Start with the Rare Initiation Codon ATT10.1128/mBio.00467-152150-7511https://doaj.org/article/3df68031778a4f8fbdf7fb021cbe20af2015-07-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00467-15https://doaj.org/toc/2150-7511ABSTRACT The endocarditis and biofilm-associated pili (Ebp) are important in Enterococcus faecalis pathogenesis, and the pilus tip, EbpA, has been shown to play a major role in pilus biogenesis, biofilm formation, and experimental infections. Based on in silico analyses, we previously predicted that ATT is the EbpA translational start codon, not the ATG codon, 120 bp downstream of ATT, which is annotated as the translational start. ATT is rarely used to initiate protein synthesis, leading to our hypothesis that this codon participates in translational regulation of Ebp production. To investigate this possibility, site-directed mutagenesis was used to introduce consecutive stop codons in place of two lysines at positions 5 and 6 from the ATT, to replace the ATT codon in situ with ATG, and then to revert this ATG to ATT; translational fusions of ebpA to lacZ were also constructed to investigate the effect of these start codons on translation. Our results showed that the annotated ATG does not start translation of EbpA, implicating ATT as the start codon; moreover, the presence of ATT, compared to the engineered ATG, resulted in significantly decreased EbpA surface display, attenuated biofilm, and reduced adherence to fibrinogen. Corroborating these findings, the translational fusion with the native ATT as the initiation codon showed significantly decreased expression of β-galactosidase compared to the construct with ATG in place of ATT. Thus, these results demonstrate that the rare initiation codon of EbpA negatively regulates EbpA surface display and negatively affects Ebp-associated functions, including biofilm and adherence to fibrinogen. IMPORTANCE Enterococcus faecalis is among the leading causes of serious infections in the hospital setting, and the endocarditis and biofilm-associated pili (Ebp) have been shown to play significant roles in E. faecalis pathogenesis. Understanding the regulation of virulence is important for the development of new approaches to counteract multidrug-resistant pathogens. We previously predicted that ATT, which has been reported to start protein synthesis only in rare instances, is the most likely translational start codon of EbpA in E. faecalis. Here, we demonstrate that ATT is the initiation codon of EbpA and, relative to a constructed ATG start codon, results in smaller amounts of EbpA on the surface of the cells, attenuating biofilm formation and fibrinogen adherence, phenotypes associated with the ability of E. faecalis to cause infections. This provides the first example of pilus regulation through the use of an ATT initiation codon.Maria Camila MontealegreSabina Leanti La RosaJung Hyeob RohBarrett R. HarveyBarbara E. MurrayAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 6, Iss 3 (2015)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Maria Camila Montealegre Sabina Leanti La Rosa Jung Hyeob Roh Barrett R. Harvey Barbara E. Murray The <named-content content-type="genus-species">Enterococcus faecalis</named-content> EbpA Pilus Protein: Attenuation of Expression, Biofilm Formation, and Adherence to Fibrinogen Start with the Rare Initiation Codon ATT
description	ABSTRACT The endocarditis and biofilm-associated pili (Ebp) are important in Enterococcus faecalis pathogenesis, and the pilus tip, EbpA, has been shown to play a major role in pilus biogenesis, biofilm formation, and experimental infections. Based on in silico analyses, we previously predicted that ATT is the EbpA translational start codon, not the ATG codon, 120 bp downstream of ATT, which is annotated as the translational start. ATT is rarely used to initiate protein synthesis, leading to our hypothesis that this codon participates in translational regulation of Ebp production. To investigate this possibility, site-directed mutagenesis was used to introduce consecutive stop codons in place of two lysines at positions 5 and 6 from the ATT, to replace the ATT codon in situ with ATG, and then to revert this ATG to ATT; translational fusions of ebpA to lacZ were also constructed to investigate the effect of these start codons on translation. Our results showed that the annotated ATG does not start translation of EbpA, implicating ATT as the start codon; moreover, the presence of ATT, compared to the engineered ATG, resulted in significantly decreased EbpA surface display, attenuated biofilm, and reduced adherence to fibrinogen. Corroborating these findings, the translational fusion with the native ATT as the initiation codon showed significantly decreased expression of β-galactosidase compared to the construct with ATG in place of ATT. Thus, these results demonstrate that the rare initiation codon of EbpA negatively regulates EbpA surface display and negatively affects Ebp-associated functions, including biofilm and adherence to fibrinogen. IMPORTANCE Enterococcus faecalis is among the leading causes of serious infections in the hospital setting, and the endocarditis and biofilm-associated pili (Ebp) have been shown to play significant roles in E. faecalis pathogenesis. Understanding the regulation of virulence is important for the development of new approaches to counteract multidrug-resistant pathogens. We previously predicted that ATT, which has been reported to start protein synthesis only in rare instances, is the most likely translational start codon of EbpA in E. faecalis. Here, we demonstrate that ATT is the initiation codon of EbpA and, relative to a constructed ATG start codon, results in smaller amounts of EbpA on the surface of the cells, attenuating biofilm formation and fibrinogen adherence, phenotypes associated with the ability of E. faecalis to cause infections. This provides the first example of pilus regulation through the use of an ATT initiation codon.
format	article
author	Maria Camila Montealegre Sabina Leanti La Rosa Jung Hyeob Roh Barrett R. Harvey Barbara E. Murray
author_facet	Maria Camila Montealegre Sabina Leanti La Rosa Jung Hyeob Roh Barrett R. Harvey Barbara E. Murray
author_sort	Maria Camila Montealegre
title	The <named-content content-type="genus-species">Enterococcus faecalis</named-content> EbpA Pilus Protein: Attenuation of Expression, Biofilm Formation, and Adherence to Fibrinogen Start with the Rare Initiation Codon ATT
title_short	The <named-content content-type="genus-species">Enterococcus faecalis</named-content> EbpA Pilus Protein: Attenuation of Expression, Biofilm Formation, and Adherence to Fibrinogen Start with the Rare Initiation Codon ATT
title_full	The <named-content content-type="genus-species">Enterococcus faecalis</named-content> EbpA Pilus Protein: Attenuation of Expression, Biofilm Formation, and Adherence to Fibrinogen Start with the Rare Initiation Codon ATT
title_fullStr	The <named-content content-type="genus-species">Enterococcus faecalis</named-content> EbpA Pilus Protein: Attenuation of Expression, Biofilm Formation, and Adherence to Fibrinogen Start with the Rare Initiation Codon ATT
title_full_unstemmed	The <named-content content-type="genus-species">Enterococcus faecalis</named-content> EbpA Pilus Protein: Attenuation of Expression, Biofilm Formation, and Adherence to Fibrinogen Start with the Rare Initiation Codon ATT
title_sort	<named-content content-type="genus-species">enterococcus faecalis</named-content> ebpa pilus protein: attenuation of expression, biofilm formation, and adherence to fibrinogen start with the rare initiation codon att
publisher	American Society for Microbiology
publishDate	2015
url	https://doaj.org/article/3df68031778a4f8fbdf7fb021cbe20af
work_keys_str_mv	AT mariacamilamontealegre thenamedcontentcontenttypegenusspeciesenterococcusfaecalisnamedcontentebpapilusproteinattenuationofexpressionbiofilmformationandadherencetofibrinogenstartwiththerareinitiationcodonatt AT sabinaleantilarosa thenamedcontentcontenttypegenusspeciesenterococcusfaecalisnamedcontentebpapilusproteinattenuationofexpressionbiofilmformationandadherencetofibrinogenstartwiththerareinitiationcodonatt AT junghyeobroh thenamedcontentcontenttypegenusspeciesenterococcusfaecalisnamedcontentebpapilusproteinattenuationofexpressionbiofilmformationandadherencetofibrinogenstartwiththerareinitiationcodonatt AT barrettrharvey thenamedcontentcontenttypegenusspeciesenterococcusfaecalisnamedcontentebpapilusproteinattenuationofexpressionbiofilmformationandadherencetofibrinogenstartwiththerareinitiationcodonatt AT barbaraemurray thenamedcontentcontenttypegenusspeciesenterococcusfaecalisnamedcontentebpapilusproteinattenuationofexpressionbiofilmformationandadherencetofibrinogenstartwiththerareinitiationcodonatt AT mariacamilamontealegre namedcontentcontenttypegenusspeciesenterococcusfaecalisnamedcontentebpapilusproteinattenuationofexpressionbiofilmformationandadherencetofibrinogenstartwiththerareinitiationcodonatt AT sabinaleantilarosa namedcontentcontenttypegenusspeciesenterococcusfaecalisnamedcontentebpapilusproteinattenuationofexpressionbiofilmformationandadherencetofibrinogenstartwiththerareinitiationcodonatt AT junghyeobroh namedcontentcontenttypegenusspeciesenterococcusfaecalisnamedcontentebpapilusproteinattenuationofexpressionbiofilmformationandadherencetofibrinogenstartwiththerareinitiationcodonatt AT barrettrharvey namedcontentcontenttypegenusspeciesenterococcusfaecalisnamedcontentebpapilusproteinattenuationofexpressionbiofilmformationandadherencetofibrinogenstartwiththerareinitiationcodonatt AT barbaraemurray namedcontentcontenttypegenusspeciesenterococcusfaecalisnamedcontentebpapilusproteinattenuationofexpressionbiofilmformationandadherencetofibrinogenstartwiththerareinitiationcodonatt
_version_	1718427453576183808

The <named-content content-type="genus-species">Enterococcus faecalis</named-content> EbpA Pilus Protein: Attenuation of Expression, Biofilm Formation, and Adherence to Fibrinogen Start with the Rare Initiation Codon ATT

Ejemplares similares